We present a novel dual redox labeling approach enabling a facile relative evaluation of protein-DNA interactions based on immunoprecipitation at magnetic beads (MBIP) with subsequent electrochemical detection. DNA probes labeled with two different electroactive markers, benzofurazane and nitrobenzene, which yield reduction peaks at distinct potentials, were synthesized using primer extension (PEX) reaction. We show that using the labeled DNA probes, specific and non-specific binding of the p53 protein can be distinguished in a simple competition binding experiment, as a strong preference of the p53 protein was observed towards DNA probes bearing a specific p53 binding site (p53CON), which is in agreement with known binding properties of the p53 protein. The p53 binding to the individual DNA probes can be modulated by specific monoclonal antibodies used for the immunoprecipitation. This approach can potentially be applied, after selection of appropriate DNA probes and monoclonal antibodies, for investigations of DNA-binding properties of other proteins and thus represents a versatile tool for studies of any DNA-binding proteins.

• Klíčová slova
• DNA modification, DNA-protein interactions, Immunoprecipitation, Redox coding, Voltammetry,
• MeSH
• DNA sondy chemie   MeSH
• DNA chemie   MeSH
• elektrochemické techniky * MeSH
• imunoprecipitace MeSH
• lidé MeSH
• molekulární struktura MeSH
• monoklonální protilátky chemie   MeSH
• nádorový supresorový protein p53 chemie   MeSH
• oxidace-redukce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA sondy MeSH
• DNA MeSH
• monoklonální protilátky MeSH
• nádorový supresorový protein p53 MeSH
• TP53 protein, human MeSH Prohlížeč

Protein hydrogen/deuterium exchange (HDX) coupled to mass spectrometry (MS) can be used to study interactions of proteins with various ligands, to describe the effects of mutations, or to reveal structural responses of proteins to different experimental conditions. It is often described as a method with virtually no limitations in terms of protein size or sample composition. While this is generally true, there are, however, ligands or buffer components that can significantly complicate the analysis. One such compound, that can make HDX-MS troublesome, is DNA. In this chapter, we will focus on the analysis of protein-DNA interactions, describe the detailed protocol, and point out ways to overcome the complications arising from the presence of DNA.

• Klíčová slova
• DNA, Hydrogen/deuterium exchange, Protein–DNA binding, Structural mass spectrometry, Transcription factor,
• MeSH
• analýza dat MeSH
• chromatografie kapalinová MeSH
• DNA vazebné proteiny chemie   metabolismus   MeSH
• DNA chemie   metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• lidé MeSH
• transkripční faktory MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• vodík/deuteriová výměna a hmotnostní spektrometrie * metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• DNA MeSH
• transkripční faktory MeSH

Characterization of protein-protein and protein-DNA interactions is critical to understand mechanisms governing the biology of cells. Here we describe optimized methods and their mutual combinations for this purpose: bimolecular fluorescence complementation (BiFC), co-immunoprecipitation (Co-IP), yeast two-hybrid systems (Y2H), and chromatin immunoprecipitation (ChIP). These improved protocols detect trimeric complexes in which two proteins of interest interact indirectly via a protein sandwiched between them. They also allow isolation of low-abundance chromatin proteins and confirmation that proteins of interest are associated with specific DNA sequences, for example telomeric tracts. Here we describe these methods and their application to map interactions of several telomere- and telomerase-associated proteins and to purify a sufficient amount of chromatin from Arabidopsis thaliana for further investigations (e.g., next-generation sequencing, hybridization).

• Klíčová slova
• Bimolecular fluorescence complementation, ChIP for low-abundance proteins, Co-immunoprecipitation, Yeast two-hybrid system,
• MeSH
• Arabidopsis genetika   metabolismus   MeSH
• buněčné jádro metabolismus   MeSH
• chromatin metabolismus   MeSH
• chromatinová imunoprecipitace metody   MeSH
• DNA rostlinná metabolismus   MeSH
• DNA vazebné proteiny izolace a purifikace   metabolismus   MeSH
• imunoprecipitace metody   MeSH
• mapování interakce mezi proteiny metody   MeSH
• optické zobrazování metody   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• techniky dvojhybridového systému MeSH
• telomerasa metabolismus   MeSH
• telomery metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromatin MeSH
• DNA rostlinná MeSH
• DNA vazebné proteiny MeSH
• proteiny huseníčku MeSH
• telomerasa MeSH

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

• MeSH
• geny p53 * MeSH
• interakční proteinové domény a motivy * MeSH
• kinetika MeSH
• konfokální mikroskopie metody   MeSH
• poškození DNA * MeSH
• Publikační typ
• audiovizuální média MeSH
• časopisecké články MeSH

GFP-like 3,5-difluoro-4-hydroxybenzylideneimidazolinone (FBI) and 3,5-bis(methoxy)-4-hydroxy-benzylideneimidazolinone (MBI) labels were attached to dCTP through a propargyl linker, and the resulting labeled nucleotides (dC(MBI)TP and dC(FBI)TP) were used for a facile enzymatic synthesis of oligonucleotide or DNA probes by polymerase-catalyzed primer extension. The MBI/FBI-labeled DNA probes exerted low fluorescence that was increased 2-3.2 times upon binding of a protein. The concept was demonstrated on sequence-specific binding of p53 to dsDNA and on nonspecific binding of single strand binding protein to an oligonucleotide. The FBI label was also used for a time-resolved experiment monitoring a single-nucleotide incorporation followed by primer extension by Vent(exo-) polymerase.

• MeSH
• benzylové sloučeniny chemie   MeSH
• DNA vazebné proteiny chemie   metabolismus   MeSH
• DNA-dependentní DNA-polymerasy chemie   metabolismus   MeSH
• DNA chemie   metabolismus   MeSH
• fluorescenční barviva chemie   MeSH
• imidazoliny MeSH
• imidazoly chemie   MeSH
• molekulární sekvence - údaje MeSH
• nukleotidy chemie   metabolismus   MeSH
• párování bází MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 3,5-bis(methoxy)-4-hydroxy-benzylideneimidazolinone MeSH Prohlížeč
• 3,5-difluoro-4-hydroxybenzylidene imidazolinone MeSH Prohlížeč
• benzylové sloučeniny MeSH
• DNA vazebné proteiny MeSH
• DNA-dependentní DNA-polymerasy MeSH
• DNA MeSH
• fluorescenční barviva MeSH
• imidazoliny MeSH
• imidazoly MeSH
• nukleotidy MeSH
• Tli polymerase MeSH Prohlížeč

Interactions between proteins, drugs, water and B-DNA minor groove have been analyzed in crystal structures of 60 protein-DNA and 14 drug-DNA complexes. It was found that only purine N3, pyrimidine O2, guanine N2 and deoxyribose O4' are involved in the interactions, and that contacts to N3 and O2 are most frequent and more polar than contacts to O4'. Many protein contacts are mediated by water, possibly to increase the DNA effective surface. Fewer water-mediated contacts are observed in drug complexes. The distributions of ligands around N3 are significantly more compact than around O2, and distributions of water molecules are the most compact. Distributions around O4' are more diffuse than for the base atoms but most distributions still have just one binding site. Ligands bind to N3 and O2 atoms in analogous positions, and simultaneous binding to N3 and N2 in guanines is extremely rare. Contacts with two consecutive nucleotides are much more frequent than base-sugar contacts within one nucleotide. The probable reason for this is the large energy of deformation of hydrogen bonds for the one nucleotide motif. Contacts of Arg, the most frequent amino acid ligand, are stereochemically indistinguishable from the binding of the remaining amino acids except asparagine (Asn) and phenylalanine (Phe). Asn and Phe bind in distinct ways, mostly to a deformed DNA, as in the complexes of TATA-box binding proteins. DNA deformation concentrates on dinucleotide regions with a distinct deformation of the delta and epsilon backbone torsion angles for the Asn and delta, epsilon, zeta and chi for the Phe-contacted regions.

• MeSH
• aminokyseliny chemie   metabolismus   MeSH
• chemické modely MeSH
• deoxyribonukleotidy chemie   metabolismus   MeSH
• DNA vazebné proteiny chemie   metabolismus   MeSH
• DNA chemie   metabolismus   MeSH
• dusík chemie   MeSH
• konformace nukleové kyseliny MeSH
• krystalografie rentgenová MeSH
• kyslík chemie   MeSH
• ligandy MeSH
• makromolekulární látky MeSH
• racionální návrh léčiv MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• voda chemie   MeSH
• vodíková vazba MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminokyseliny MeSH
• deoxyribonukleotidy MeSH
• DNA vazebné proteiny MeSH
• DNA MeSH
• dusík MeSH
• kyslík MeSH
• ligandy MeSH
• makromolekulární látky MeSH
• voda MeSH

p53 plays critical roles in regulating cell cycle, apoptosis, senescence and metabolism and is commonly mutated in human cancer. These roles are achieved by interaction with other proteins, but particularly by interaction with DNA. As a transcription factor, p53 is well known to bind consensus target sequences in linear B-DNA. Recent findings indicate that p53 binds with higher affinity to target sequences that form cruciform DNA structure. Moreover, p53 binds very tightly to non-B DNA structures and local DNA structures are increasingly recognized to influence the activity of wild-type and mutant p53. Apart from cruciform structures, p53 binds to quadruplex DNA, triplex DNA, DNA loops, bulged DNA and hemicatenane DNA. In this review, we describe local DNA structures and summarize information about interactions of p53 with these structural DNA motifs. These recent data provide important insights into the complexity of the p53 pathway and the functional consequences of wild-type and mutant p53 activation in normal and tumor cells.

• Klíčová slova
• local DNA structures, p53 protein, protein-DNA interactions,
• MeSH
• B-DNA MeSH
• DNA chemie   genetika   metabolismus   MeSH
• konformace nukleové kyseliny * MeSH
• lidé MeSH
• nádorový supresorový protein p53 chemie   metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• B-DNA MeSH
• DNA MeSH
• nádorový supresorový protein p53 MeSH
• triplex DNA MeSH Prohlížeč

In view of the importance of protein-DNA interactions in biological processes, we extracted from the Protein Data Bank several one-to-one complexes of amino acids with nucleotides that matched certain geometric and energetic specificity criteria and investigated them using quantum chemistry methods. The CCSD(T)/CBS interaction energies were used as a benchmark to compare the performance of the MP2.5, MP2-F12, DFT-D3, and PM6-D3H4 methods. All methods yielded good agreement with the reference values, with declining accuracy from MP2.5 to PM6-D3H4. Regardless of the site of interaction, the minima found after full optimization in implicit solvent with high dielectric constant were close to the structures experimentally detected in protein-DNA complexes. According to DFT-SAPT analysis, the nature of noncovalent interactions strongly depends on the type of amino acid. The negatively charged sugar-phosphate backbone of DNA heavily influences the strength of interactions and must be included in the computational model, especially in the case of interactions with charged amino acids.

• MeSH
• aminokyselinové motivy MeSH
• databáze proteinů MeSH
• DNA metabolismus   MeSH
• proteiny chemie   metabolismus   MeSH
• substrátová specifita MeSH
• termodynamika MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA MeSH
• proteiny MeSH

A series of 7-[4-(trifluoroacetyl)phenyl]-7-deazaadenine and -7-deazaguanine as well as 5-substituted uracil and cytosine 2'-deoxyribonucleosides and mono- and triphosphates were synthesized through aqueous Suzuki-Miyaura crosscoupling of halogenated nucleosides or nucleotides with 4-(trifluoroacetyl)phenylboronic acid. The modified nucleoside triphosphates were good substrates for DNA polymerases applicable in primer extension or PCR synthesis of modified oligonucleotides or DNA. Attempted cross-linking with a serine-containing protein did not proceed, however the trifluoroacetophenone group was a sensitive probe for the study of DNA-protein interactions by 19F NMR.

• MeSH
• acetofenony chemická syntéza   chemie   MeSH
• DNA chemie   MeSH
• fluor MeSH
• magnetická rezonanční spektroskopie MeSH
• molekulární struktura MeSH
• oligonukleotidy chemie   MeSH
• proteiny chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• acetofenony MeSH
• DNA MeSH
• fluor MeSH
• oligonukleotidy MeSH
• proteiny MeSH

Interaction with the DNA minor groove is a significant contributor to specific sequence recognition in selected families of DNA-binding proteins. Based on a statistical analysis of 3D structures of protein-DNA complexes, we propose that distortion of the DNA minor groove resulting from interactions with hydrophobic amino acid residues is a universal element of protein-DNA recognition. We provide evidence to support this by associating each DNA minor groove-binding amino acid residue with the local dimensions of the DNA double helix using a novel algorithm. The widened DNA minor grooves are associated with high GC content. However, some AT-rich sequences contacted by hydrophobic amino acids (e.g., phenylalanine) display extreme values of minor groove width as well. For a number of hydrophobic amino acids, distinct secondary structure preferences could be identified for residues interacting with the widened DNA minor groove. These results hold even after discarding the most populous families of minor groove-binding proteins.

• Klíčová slova
• DNA shape, hydrophobic, indirect readout, minor groove, protein–DNA interaction, specific recognition,
• MeSH
• algoritmy MeSH
• aminokyselinové motivy MeSH
• aminokyseliny chemie   MeSH
• Arabidopsis metabolismus   MeSH
• DNA vazebné proteiny metabolismus   MeSH
• DNA chemie   MeSH
• fenylalanin chemie   MeSH
• hydrofobní a hydrofilní interakce * MeSH
• konformace nukleové kyseliny MeSH
• kyselina glutamová chemie   MeSH
• lidé MeSH
• proteiny chemie   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• sekundární struktura proteinů MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aminokyseliny MeSH
• DNA vazebné proteiny MeSH
• DNA MeSH
• fenylalanin MeSH
• kyselina glutamová MeSH
• proteiny MeSH